CDI molecules present various kinds of lipid antigens to T lymphocytes. Human CD1a, CD1b, and CD1c could play an important role in host defense by presenting lipids from microbes. CD1d, by contrast, stimulates a subpopulation of immune regulatory T lymphocytes called NK T cells. NK T cells have been implicated in the immune response to tumors and in the prevention of autoimmune disease. Autologous lipids may be involved in the CD1d-mediated stimulation of NK T cells; they also react to the synthetic lipoglycan, alpha-galactosyl ceramide. Relatively little is known regarding the pathway for the CD1-mediated presentation of lipids. Here we propose a comprehensive study of this pathway, using the NK T cell recognition of alphaGalCer plus mCD1d. We will analyze the intracellular trafficking of antigens and mCD1d molecules, lipoglycan antigen processing, and the in vivo affects on NK T cells of altering the antigen processing pathway or the trafficking of mCD1d. Preliminary data indicate that a disaccharide analog of GalCer requires processing. In the first aim, we will confirm this biochemically, and test a variety of analogs with different carbohydrate and lipid moieties, to determine whether lipid antigen processing is widespread and versatile. In the second aim, we will express mutant mCD1d molecules with restricted intracellular trafficking, and carry out cell fractionation studies to determine where processing and mCD1 antigen binding occur. In the third aim, we will determine the in vivo effect of altering mCD1d intracellular traffic on NK T cell development and function. In the fourth aim, we will explore how alterations in carbohydrate due to deficiency of the enzyme alpha-galactosidase A lead to in vivo deficits in NK T cell number and function. The results from these studies will aid in our understanding of lipid antigen presentation, and they should help in the design of lipid antigen-based vaccines and in attempts to alter immune regulation by stimulating NK T cells.